Abstract
Cancer-specific metabolic alterations are of high interest as therapeutic targets. These alterations vary between tumor types, and to employ metabolic targeting to its fullest potential there is a need for robust methods that identify candidate targetable metabolic pathways in individual cancers. Currently, such methods include 13C-tracing studies and mass spectrometry/ magnetic resonance spectroscopic imaging. Due to high cost and complexity, such studies are restricted to a research setting. We here present the validation of a novel technique of metabolic profiling, based on multiplex targeted next generation sequencing of RNA with single molecule molecular inversion probes (smMIPs), designed to measure activity of and mutations in genes that encode metabolic enzymes. We here profiled an isogenic pair of cell lines, differing in expression of the Von Hippel Lindau protein, an important regulator of hypoxia-inducible genes. We show that smMIP-profiling provides relevant information on active metabolic pathways. Because smMIP-based targeted RNAseq is cost-effective and can be applied in a medium high-throughput setting (200 samples can be profiled simultaneously in one next generation sequencing run) it is a highly interesting approach for profiling of the activity of genes of interest, including those regulating metabolism, in a routine patient care setting.
Highlights
Cancer-specific metabolic alterations are of high interest as therapeutic targets
Clear cell renal cell carcinoma is considered a metabolic cancer with metabolic alterations resulting from inactivating mutations in or epigenetic silencing of VHL found in ~80% of cases38, 39. pVHL is a major regulator of ubiquitination and breakdown of hypoxia inducible transcription factors HIF-1α and HIF-2α38
To investigate whether single molecule molecular inversion probes (smMIPs) can be used for multiplex determination of gene expression levels, concomitant with variant detection, we designed a smMIP set for targeted detection and sequencing of 104 transcripts encoding metabolic enzymes and 18 tyrosine kinases with relevance for oncology
Summary
Cancer-specific metabolic alterations are of high interest as therapeutic targets These alterations vary between tumor types, and to employ metabolic targeting to its fullest potential there is a need for robust methods that identify candidate targetable metabolic pathways in individual cancers. Such methods include 13C-tracing studies and mass spectrometry/ magnetic resonance spectroscopic imaging. Because smMIP-based targeted RNAseq is cost-effective and can be applied in a medium high-throughput setting (200 samples can be profiled simultaneously in one generation sequencing run) it is a highly interesting approach for profiling of the activity of genes of interest, including those regulating metabolism, in a routine patient care setting. Mutations in the aforementioned metabolic enzymes and in VHL have been shown to induce epigenetic alterations that affect expression of other metabolic enzymes in an unpredictable fashion[40,41,42,43]
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